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Chapter I
10
2.0 CHAPTER – I SYNTHESIS, CARDIAC EFFECTS AND ANTI BACTERIAL ACTIVITY OF 3, 4 - DIHYDROPYRIMIDIN - 2 - (1H) - ONE - 5 CARBOXYLATES 2.1 INTRODUCTION
Biginelli compounds or 3,4-Dihydropyrimidin-2(1H)-ones (DHPMs) possess
interesting biological applications (Kappe, 1993; Snider and Shi, 1993; Overman et al.,
1995; Kappe, 2000a; 2000b). The untapped potentials of DHPMs were explored because
of their apparent structural similarities with clinically important dihydropyridines (DHPs)
of nifedipine-type (Rovnyak et al., 1995). This reason accounts for the increased number
of publications and patents in the DHPM synthesis. Synthesis, reactions and medicinal
applications of DHPMs have been extensively reviewed (Kappe, 1993; 1998; 2000b;
Kappe and Stadler, 2004). As early as 1930s, its simple derivative, 4-chlorophenyl-2-
thio-DHPM was patented as an agent for the protection of wool against moths (Ertan et
al., 1933). Later, the interest has focussed on the antiviral activity of Biginelli
compounds, eventually leading to the excellent antiviral agent, nitractin (Hurst and Ann,
1962). The very same compound also exhibits modest antibacterial activity.
2.1.1 Synthetic Approaches
2.1.1.1 Biginelli Reaction
The Biginelli reaction is a multiple-component reaction invented by Pietro
Biginelli in 1891 that creates 3,4-dihydropyrimidin-2(1H)-ones by the condensation of a
dicarbonyl compound , an aryl aldehyde and urea (Biginelli, 1891; Biginelli, 1893; Zaugg
and Martin, 1965; Kappe, 1993).
Chapter I
11
O
H
O
O O
NH2 NH2
O
N
NH
O
O
O
H
++
Acidified alcohol
Reflux
Ranu et al. (2000) have described the indium (III) chloride catalyzed synthesis of
dihydropyrimidinones
NH2 NH2
X O O
R1 R2
N
NH
XR1
O R3
R2
H
R3
CHO + +
X = O, S
THF
InCl3
Ma et al. (2000) have reported the lanthanide triflate catalyzed Biginelli reaction.
NH2 NH2
O O O
OEt
CHO
N
NH
O
O
EtO
H
+ + + H2OLn(OTf)3
Lu and Ma (2000) have reported the iron (III)-catalyzed synthesis of
dihydropyrimidinones.
NH2 NH2
O O O
OEt
CHO
N
NH
O
O
EtO
H
+ + + H2OIron III
Ananda Kumar et al. (2001) have reported the Mn (OAc) 3 catalyzed synthesis of
3,4-dihydropyrimidin-2(1H)-ones by refluxing in acetonitrile for 2-4 h.
Chapter I
12
NH2 NH2
O
O O
OEt
N
NH
O
O
EtO
H
CHO
+Mn(OAc)3. 2H2O
CH3CN
Sabitha et al. (2003) have reported the vanadium III chloride catalyzed Biginelli
condensation.
O
ONH2
NH2
X
H O
R2
R1
+
R
reflux, 2 h heat
VCl3/CH3CN
N
NH
O
XH
R
R1
R2
Kappe and Falsone (1998) have reported the polyphosphate ester-mediated
synthesis of dihydropyrimidines.
NH2 NH2
X O O
R1 R2
N
NH
XR1
O R3
R2
H
R3
CHO + +
X = O, S
PPE
Salehi et al. (2003) have reported the silica sulfuric acid catalyzed synthesis of
3,4-dihydropyrimidin-2(1H)-ones by refluxing in ethanol for 6 h.
NH2 NH2
O
O O
OEt
N
NH
O
O
EtO
H
CHO
+ Silica Sulfuric Acid
EtOH, 6 h, heat
Chapter I
13
Rani et al. (2001) have reported the zeolite-catalyzed cyclocondensation reaction
for the synthesis of 3,4-dihydropyrimidin-2(1H)-ones.
NH2 NH2
O O O
OEtN
NH
O
O R
EtO
H
R
CHO + +Zeolite
Toluene, Reflux
Huang et al. (2005) have achieved the highly enantioselective synthesis of
dihydropyrimidines by using a chiral ytterbium catalyst.
NH2 NH2
O O O
OEtN
NH
O
O
EtO
H
CHO
+ +Ligand, Ln(OTf)3
N N
OH OH
R
R
R
R
N NPh Ph
R = H, tBu
Ligand =
Peng and Deng (2001) have reported the ionic liquid mediated Biginelli synthesis
of 3,4-dihydropyrimidin-2(1H)-ones at 100 ºC.
R CHO NH2 NH2
O
R1 R2
O O
N
NH
R
R1 O
O
H
R2+ +Ionic liquid
100 ºC
Chapter I
14
Debache et al. (2006) have reported the phenyl boronic acid catalyzed Biginelli
reaction in acetonitrile under reflux condition.
NH2 NH2
O
O O
OEt
N
NH
O
O
EtO
H
CHO
+PhB(OH)2
CH3CN
2.1.1.2 Micro wave-assisted organic reactions
The rapid heating associated with microwave technology has been applied in a
number of disciplines such as the preparation of samples for analysis, application to
waste treatment, polymer technology, drug release or targeting and hydrolysis of proteins
and peptides. Its application in organic synthesis has not only reduced the reaction time in
many folds but also has proven to give better yields.
The practical applications of the various methods under conventional thermal
conditions suffer from the disadvantages such as the use of expensive or less readily
available reagents, vigorous reaction conditions, prolonged reaction conditions and
tedious manipulations in the isolation of the pure products. These necessitate a method
for versatile, simple and environmentally friendly process whereby compounds may be
obtained under microwave assisted milder conditions (Gohain et al., 2004).
2.1.1.3 Enantiomerically pure dihydropyrimidines
Dihydropyrimidines of the Biginelli type are inherently asymmetric molecules,
and therefore are usually obtained as racemic mixtures. The influence of the absolute
configuration at the stereogenic C-4 on biological activity is well documented (Atwal et
al., 1990a, 1990b). Access to enantiomerically pure DHPMs is therefore of considerable
interest and a prerequisite for the development of drugs in this field (Atwal et al., 1991).
Chapter I
15
In the absence of any known general asymmetric synthesis for this heterocyclic target
system, resolution strategies have so far been the method of choice to obtain
enantiomerically pure DHPMs.
Due to recent advances in preparative chromatographic enantioseparation
N
NH
MeMe
O
EtO
O
N
NH
Me
O
EtO
OBr
P ON
O H
P ON
O N
NH
Me
O
EtO
O
Br2, CHCl 3
CSP
Equation 1: Preparation of Chiral Stationary Phase (CSP) DHPMs by attaching macroporous
aminomethacrylate on DHPM scaffolds.
techniques, enantioselective HPLC and related methods have gained importance in the
synthesis of single-enantiomer drugs and intermediates. In a recent study by Kleidernigg
and Kappe (1997), it is reported that the chromatographic enantioseparation of DHPM
derivatives can be accomplished by using a variety of commercially available chiral
stationary phases (CSPs) in normal and reversed phase analytical HPLC. Resolution of
the enantiomers of DHPMs using semipreparative chiral HPLC, followed by attaching to
monodisperse macroporous aminomethacrylate beads (Equation 1) provided the novel
polymer-based CSP.
Such designer “CSPs” would prove extremely useful for the efficient separation
of not only DHPMs but other structurally related compounds as well. The chiral
separation of DHPMs by capillary electrophoresis (CE) using quarternary ammonium-β-
cyclodextrin as chiral buffer additive has also been reported (Wang et al., 2000).
Biocatalytic strategy towards the preparation of enantiopure (R) - and (S) - 32926
Chapter I
16
Equation 2: Biocatalytic strategy of synthesizing optically pure DHPM, (R)-32926, an antihypertensive agent via enzymatic resolution mediated by Thermomyces lanuginosus lipase.
has been developed. The key step in the synthesis is the enzymatic resolution of an N-3-
acetoxymethyl-activated dihydropyrimidinone precursor by Thermomyces lanuginosus
lipase. Readily available racemic DHPM was hydroxymethylated at N-3 with
formaldehyde, followed by standard acetylation with acetyl chloride. The resulting N-3-
acetoxy methyl-activated DHPM was then cleaved enantioselectively by Thermomyces
lanuginosus lipase with excellent selectivity (Equation 2). Degradation of unreacted 3-
hydroxy methylated (R)-DHPM with aqueous ammonia produced (R)-DHPM, which was
converted into the desired target structure, (R)-32926, in one step by N-3 carbomoylation
with trichloroacetyl isocyanate (Stadler and Kappe, 2000).
A critical point in every preparation of enantiomerically pure materials, regardless
of the method, is the assignment of absolute configuration. For the DHPM series, a
simple protocol for absolute configuration assignment based on the combination of
enantioselective HPLC and circular dichroism (CD) spectroscopy has been developed.
NH
NH
O
i-PrO
O
NO2
NH
N
O
i-PrO
O
NO2
OAc
NH
NH
O
i-PrO
O
NO2
CCl3
O
OCN
NH
N
O
i-PrO
O
NO2
NH2
O
1. CH2O
2. AcCl
1. Lipase
2. AgNO3
(R) 32926
(R)
Chapter I
17
By the comparison of the characteristic CD spectra of individual DHPM enantiomers
with reference samples of known absolute configuration, the absolute configuration of 4-
aryl DHPMs could be established. The enantiomers were obtained by semipreparative
HPLC separation of racemic DHPMs on chiral stationary phases. The characteristic CD
activity of the enamide chromophore around 300 nm allows the assignment of absolute
configuration in this series of dihydropyrimidine derivatives (Krenn et al., 1999).
2.1.2 Biological activities
Biginelli compounds show a diverse range of biological activities. As early as
1930, these derivatives were patented as agents for the protection of wool against moths
(Folkers and Johnson, 1933). In 1893, the synthesis of functionalized DHPMs was
reported for the first time by Biginelli. This efficient approach to partly reduced
pyrimidines was ignored in the following decades and therefore, the pharmacological
properties of this interesting heterocyclic scaffolds remained unexplored. Since the early
1980s, however, interest in DHPMs has increased significantly (Kappe, 1993). This was
originally due to the apparent structural similarity of DHPMs to the well known
dihydropyridines calcium channel modulators of the Hantzsch type (Atwal et al., 1990a).
It was soon established that DHPMs exhibit a similar pharmacological profile to DHP
calcium channel modulators of the nifedipine type and much activity has been observed
in this area (Cho et al., 1989; Schnell et al., 2000). They are also α-1a adrenoreceptor
selective antagonists useful for the treatment of benign prosthetic hyperplasia (Wetzel et
al., 1995). The recent identification of a DHPM analog as potential new anticancer lead
that is involved in blocking mitosis by inhibition of a kinesin motor protein is another
biological activity (Mayer et al., 1999).
Chapter I
18
2.1.2.1 Calcium channel modulators
DHPs e.g., nifedipine are the most studied class of organic calcium channel
modulators. More than 25 years after the introduction of nifedipine, many DHP analogs
have now been synthesised and numerous second-generation commercial products have
appeared on the market e.g. nicardipine, amlodipine, felodipine, etc. (Bossert and Vater,
1989). These substances act by inhibiting the entry of Ca2+ into
ONH
N
Me
O
MeO ON
ONO2
NH
Me Me
OO
OMeMeO
NO2
(a) (b)
ONH
N
Me
OCOOC7H15-n
O2N
O
ONH
N
Me
O
COOEt
O2N
EtO
SNH
N
Me
O
COOEt
O2N
OiPr
(c) (d) (e)
Fig. 1.1a-e Structures of DHPM calcium channel modulators
the cells of cardiac and vascular muscle through the voltage-dependent calcium channels
and decrease ventricular contractility by the same mechanism. The cardiovascular
activity of DHPMs was first recognized by Khanina et al. (1978) who reported on β-
aminoethyl esters of DHPMs that exhibit moderate hypotensive activity and coronary
dilatory properties. Difluoromethoxy-substituted analogs of DHPMs also showed similar
levels of activity (Kastron et al., 1987). DHPMs were shown to be potent calcium
channel blockers; however most did not show significant antihypertensive activity in vivo
Chapter I
19
(Atwal et al., 1990a). Further structural modifications on the dihydropyrimidine ring led
to DHPMs bearing an ester group at N 3 (Atwal et al., 1990b) thereby more closely
resembling nifedipine-type dihydropyrimidines (Fig. 1.1).
Among the most potent derivatives in a series of N (3) - substituted DHPMs, is
the thiourea derivative (Fig. 1.1(e)) (Atwal et al., 1990b). These are calcium channel
blockers devoid of antihypertensive activity in vivo. The lack of oral activity of these
derivatives has been rationalized by their rapid metabolism. Further modification of the
substituent at N (3) - subsequently led to the development of orally active long-lasting
hypertensive agents such as SQ 32926 and SQ 32547 (Fig. 1.2). The improved oral
bioavailability results from the increased chemical stability of the urea functionality. Both
compounds have anti-ischaemic properties in animal models (Grover et al., 1995). Of
critical importance for the biological activity of most of the DHPMs shown in Fig. 1.3 is
the absolute stereochemistry at the C4 stereocenter.Pharmacologic studies with resolved
enantiomers have demonstrated that, for example, for DHPM (Fig. 1.3(e)) a > 1000 –fold
difference in potency can be observed (Atwal et al., 1990b). For the orally active
antihypertensive DHPM derivatives SQ 32926 and SQ 32547, it was established that the
desired antihypertensive effect resides solely in the (R)–enantiomer (Atwal et al., 1991;
Rovnyak et al., 1992).
SNH
N
Me
O
O2N
O CONH2iPr
SQ 32926
N
ONH
N
Me
O
O
NO2
O
OF
iPr
SQ 32547
Fig. 1.2 Structures of DHPM based calcium channel modulators
Chapter I
20
2.1.2.2 α1a- Adrenergic receptor antagonists
Benign prosthetic hyperplasia (BPH) is a progressive enlargement of the prostate
resulting in a number of obstructive and irritative symptoms. The incidence of BHP
increases with advancing age, such that 70 % of males, over 70 years old, manifest its
symptoms. Non-selective α1a-adrenoreceptor antagonists, e.g. terazosin, are currently
being approved pharmaceuticals for treating BPH (Nagarathnam et al., 1998). It is
reported that the functional potency of a number of α1 antagonists correlates well with
the binding affinity for α1a subtype at the cloned human receptors. Thus the DHPM
scaffold SNAP 6201 was developed with good binding affinity and excellent subtype
selectivity for the α1a receptor with no cardiovascular effects (Fig. 1.3).
N
Ph
COOH
ONH
N
Et
O
NH2
O
FF
NH
SNAP 6201
Fig. 1.3 α1a Subtype cloned human receptors.
2.1.2.3 Mitotic kinesin inhibitors
A common strategy for cancer therapy is the development of drugs that interrupt
the cell cycle during the mitosis stage. Compounds that perturb microtubule shortening
(depolarization) or lengthening (polymerization) cause arrest of the cell cycle in mitosis
due to perturbation of the normal microtubule dynamics necessary for chromosome
movement (Atwal and Moreland, 1991). A variety of such drugs that bind to tubulin and
thus inhibit spindle assembly are currently used in cancer therapy e.g. paclitaxel and
Chapter I
21
docetaxel (Jordan et al., 1998). Mayer et al. (1999) have recently identified the
structurally rather simple DHPM - termed monastrol - as a novel cell-permeable
molecule, that blocks normal bipolar mitotic spindle assembly in mammalian cells and
therefore, causes cell cycle arrest. Monastrol (Fig. 1.4) blocks mitosis by specifically
inhibiting the motor activity of the mitotic kinesin Eg5, a motor protein required for
spindle bipolarity. Monastrol is the only cell- permeable molecule currently known to
specifically inhibit mitotic kinesin Eg5 and can therefore be considered as a lead for the
development of new anticancer drugs.
Fig. 1.4 Monastrol, the only cell-permeable molecule inhibiting mitotic kinesin Eg5
2.1.2.4 Miscellaneous biological effects
As early as the 1940s, DHPM type of compounds (Fig. 1.5) was shown to possess
antiviral activity. Eventually, the nitrofuryl-substituted analog nitractin (Fig. 1.5a) was
developed which displayed good activity against the viruses of the trachoma group, in
addition to showing modest antibacterial activity. Other structurally simple DHPMs were
screened as anti-tumor agents and found to be active against, for example, Walker
carcinosarcoma in rats and mice. Pyrimidine-5-carboxamides type were claimed to have
anticarcinogenic activity, while other derivatives were reported to have blood platelet
aggregation inhibitory activity (Tozkoparan et al., 1995), or were shown to inhibit the
uptake of adenosine by thrombocytes (Fig. 1.5b). Fused DHPMs, such as thiazolo [3,2-a]
– pyrimidine (Fig. 1.5c) and pyrimido [2,1-b] [1,3] thiazine (Fig. 1.5d) were reported to
SNH
NH
Me
O
OH
EtO
Chapter I
22
have anti-inflammatory activity. (Tozkoparan et al., 1999). Fungicidal activity towards
Aspergillus niger and A. ochraceus was demonstrated for simple 2-thioxo DHPMs.
Not only synthetic DHPM derivatives, but also several natural products with
interesting biological activities containing the dihydropyrimidine-5-carboxylate core,have
been isolated from marine sources.Among these,the batzelladine alkaloids A and B,
inhibits the binding of HIV envelop protein gp-120 to human CD4 cells and therefore,are
potential new leads for AIDS therapy (Heys et al., 2000).
(a) (b) (c) (d) Fig. 1.5a-d Structures of biologically active DHPMs
Extensive literature survey reveals that there are no reports on the cardiotonic and
antibacterial activity of DHPMs. Therefore in the present study we describe the synthesis,
cardiovascular and antibacterial activity of DHPMs.
Cardiovascular diseases have been the principal cause of death in many
developing countries (WHO, 1986) and disability inindustrialized nations and is among
the syndromes most commonly encountered in clinical practice (Seth, 1999). The
diagnosis of heart failure carries a risk of mortality comparableto that of the major
malignancies (Henry and Colucci, 2001). Heart failure occurs when cardiac output is
insufficient to meet the demands of tissue perfusion and may primarily be due to systolic
or diastolic dysfunction (Tripathi, 2003). It is frequently, but not always, caused by a
NH
NH
Me S
O
NH2
NMe
O
NH2 N
S
OMe
NMe
O
NS
O
EtO
O
ONH
NH
Me
O
EtO
O2N
Chapter I
23
defect in myocardial contraction (Braunwald, 1988). Myocardial contractility is largely
dependent upon the activity of the cardiac sympathetic nerves, but it can also be
increased by circulating catecholamines, tachycardia, and inotropic drugs (Julien et al.,
1998). These drugs induce changes in myoplasmic calcium and this may be responsible
for the cardioactive properties. Cardiac glycosides and catecholamines have been used as
the main therapeutic drugs in the treatment of congestive cardiac failure (Kitada et al.,
1987). However, the dangers of cardiac glycosides intoxication are well documented
(Beller et al., 1971) and doubts have been expressed about their long-term effectiveness.
The use of catecholamines is limited by their insufficient differentiation between positive
inotropic and chronotropic actions, their potential arrhythmogenic properties, and
tachyphylaxis due to receptor downregulation (Kitada et al., 1987).
3,4-Dihydropyrimidinones (DHPMs) also called Biginelli compounds possess
interesting biological applications. The apparent structural similarities of DHPMs to the
well-known Hantzsch-type dihydropyridines, calcium channel modulators, suggest a
good scope for this class of compounds in the field of medicinal chemistry (Rovnyak et
al., 1992 & 1995). Calcium channel blockers are used in the treatment of angina,
hypertension, cardiac arrhythmias (Jacob, 1992) and have a limited role in heart failure.
The dihydropyridines are the most potent Ca2+ channel blockers. They have little effect
on the myocardium and conducting tissue. Used alone, they often cause a reflex
tachycardia which can be avoided by concomitant use of a β-blocker. More than 25 years
after the introduction of nifedipine, many DHP analogs and numerous second generation
products are available in the market (e.g., nicardipine, amlodipine, felodipine, etc.).
Advances in the knowledge of the biochemical and physiological changes during cardiac
Chapter I
24
failure as well as the development of new diagnostic and surgical procedures in
cardiovascular medicines have been remarkable in the last few decades. Unfortunately no
such claim could be made with respect to the development of new pharmacological
agents with clinically useful positive inotropic properties (Vasavada et al., 1990). Nearly
two centuries have passed since William Withering described the cardiovascular effects
of Digitalis purpurea in 1785; however the basic treatment of congestive heart failure
still depends on the cardiac glycosides. Although hundreds of cardiac glycosides have
been investigated, not one has been found with a wide therapeutic index. This
necessitates research for new drugs, which increase cardiac muscle contractility with a
broad therapeutic index. It is well-established that only one particular enantiomer is
responsible for calcium channel antagonist activity between the existing two
enantiomeric DHPMs in the racemic mixture (Goldman et al., 1991; Kappe, 2000b).
Herein, for the first time, the cardiotonic activity of DHPMs on an isolated perfused frog
heart was demonstrated. The action may be attributed to the presence of the more potent
agonistic enantiomer in the racemic mixture. Further in this study, synthesis,
cardiovascular and antibacterial activity of DHPMs, which is vital for progress in the
medical field were described.
Chapter I
25
2.2 OBJECTIVES
DHPM scaffolds are exhibiting wide range of biological and medicinal activities,
and synthetic procedures to prepare them are increasing tremendously. It shows a broad
range of biological activity like calcium channel blockers, antihypertensive agents and
α1a-antagonists, antiviral, antibacterial and anti-inflammatory activities.
The aim of the present work was to study
Synthesis of structurally challenging DHPMs through improved protocols
such as application of novel catalysts, microwave irradiation and neat
synthesis.
Evaluation of the possible cardiac and antibacterial activity of the
synthesized and structurally characterized DHPMs.
Chapter I
26
2.3 EXPERIMENTAL
2.3.1 Equipments
Melting points were determined in open capillary tubes and are uncorrected. IR
measurements were obtained as KBr pellets using Perkin-Elmer spectrum RXI FT-IR.
The 1H NMR and 13C NMR spectra were recorded in CDCl3 + DMSO-d6 with JEOL 400
MHz (model GSX 400) high resolution NMR spectrometer with TMS as internal
standard. Mass spectra were obtained using JEOL DX-303 in EI ionization mode at 70
eV. The reaction was carried out in a BPL—SANYO domestic microwave oven
operating at 2.45 GHz. TLC was performed on precoated Polygram SIL G/UV254 sheets.
The elemental analyses of the compounds were recorded using ThermoFinnigan FLASH
EA 1112 CHNS analyzer.Column chromatography was carried out using 100-200 mesh
silica gel. Ethylacetoacetate, methylacetoacetate, benzyl acetoacetate, urea, ortho-chloro
benzaldehyde, ortho-nitro benzaldehyde, anisaldehyde, Meta-and para-hydroxy
benzaldehydes were purchased from s.d.fine chemicals. Biphenyl carboxaldehyde and
isobutyrylaldehyde was purchased from Aldrich Sigma Chemicals.
2.3.2 General Procedure for the Synthesis of 4-(substituted)-3,4-
dihydropyrimidinones
A mixture of β-keto ester 1 (1 mmol), aldehyde 2 (1 mmol), and urea 3 (1 mmol)
was irradiated in acetic acid medium with few drops of concentrated HCl. The solution
was kept in an alumina bath and irradiated in a domestic microwave oven for 5–7 min
with a pulse rate of 40 sec and 30% of power. The total consumption of aldehyde as
monitored by TLC was an indication of completion of the reaction. After the reaction was
over, the mixture was poured into 150 ml of ice-cold water and heated over a water bath
Chapter I
27
for 30 min followed by stirring for 1 h at room temperature. The solid thus obtained 4a–i
was collected by filtration and column chromatographed with 1:3 petroleum ether and
ethyl acetate mixture. Characterization of the compounds by IR, 1H NMR, 13C NMR,
mass spectroscopy, elemental analyses, and melting point confirms the formation of
products.
2.3.3 Cardiovascular activity
2.3.3.1 Animals
Common Indian adult frogs of Rana tigrina species were used in the present
study. The great advantage of frog tissues is that they can function as isolated
preparations for many hours when handled carefully and to maintain the tissue
preparation, no extra supply of oxygen is needed as the frog muscles can directly imbibe
oxygen from the atmosphere. The animals were maintained as per the norms of
Committee for the Purpose of Control and Supervision of Experiments on Animal
(CPCSEA) and this experiment was cleared by CPCSEA and Institutional Animal Ethics
Committee constituted for the purpose (IAEC/SRMC&RI/14.8.2003).
2.3.3.2 Introduction
Heart diseases are prime culprits in modern world for maximum number of deaths
in Asian, American as well as European countries. Cardiac failure denotes the presence
of one of the complexes of symptoms and signs associated with the congestion of tissues
and organs or attributable to the inadequate perfusion of tissues and organs.
Cardiovascular agents are the drugs which modify the functions of cardiovascular system.
Chapter I
28
2.3.3.3 Principle
Drugs may influence the rate (chronotropy) and force (inotropy) of contraction of
the heart.An increase in the heart rate is called a ‘positive chronotropic’ response, while a
‘negative chronotropic’ response decrease in the heart rate.Similarly, an increase in the
force of contraction is called a ‘positive inotropic’ response and a decrease in the force of
contraction is called a ‘negative inotropic’ response.
2.3.3.4 Materials Requirement
Myograph board, dissection apparatus, starling heart lever, thread, pin,
muscle/force transducer (INCO) and Student’s physiograph single channel (INCO). Frog
ringer with the following composition was used as the perfusion fluid NaCl, 110.0; KCl,
1.90; CaCl2, 1.10; NaH2PO4, 0.06; NaHCO3, 2.40; dextrose, 11.10 l μM made up to 1000
ml with distilled water and sodium carboxy methyl cellulose (Loba Chemie, Mumbai).
2.3.3.5 Preparation of drug solution
The marketed digoxin tablets (Lanoxin) were obtained from the local market and
different dilutions were made with distilled water to get 25, 50 and 500 µg/ ml.Adrenalin,
verapamil (calaptin) ,metoprolol (Metocard) were obtained from the local market
and diluted with frog ringers solution to get 5 µg/ml concentration.
2.3.3.6 Preparation of test compounds
The compounds 4(a-i) were made as an aqueous suspension in 1% sodium
carboxy methyl cellulose to get 5, 50,100, 500 and 1000 μg concentration.
Chapter I
29
2.3.3.7 Method
Common Indian frogs of Rana tigrina were divided into 10 groups comprising of
standard and test compounds (4a-4i) of 6 animals each. Frogs were dissected from ventral
surface to expose the heart and pericardium was removed. The frog heart was isolated
and perfusion was done by Bulbring’s method as described by Burn (1952). The apex of
ventricle was attached to the sterling heart lever, which in turn was attached by a thread
to the muscle forced transducer and this is connected to a ‘physiograph’. Normal heart
rate, contractile amplitude was recorded. The cardiac output from the isolated frog heart
was collected every minute and measured. The various concentrations of compounds to
be tested were prepared using 1% sodium carboxy methyl cellulose and administered to
the ringer flowing through the cannula and the effects were recorded at different dose
level (5 μg, 50 μg, 100 μg, 500 μg, and 1 mg/ml). Only one compound was tested in each
preparation. The responses of the compounds were compared to those of digoxin. During
the interaction studies verapamil (5 μg/ml) in frog ringer’s solution, metoprolol, a
β-adrenergic blocker (5 μg/ml) in frog ringer’s solution and adrenaline (5 μg/ml) were
administered to the ringer solution and recordings were noted. The data presented in
figures are mean ± SEM. One-way ANOVA was used for statistical analysis. P values
< 0.01 were considered to be statistically significant.
2.3.4 Antibacterial activity 2.3.4.1 Introduction
Antibacterial activity is measured in vitro in order to determine 1) the potency of
an antibacterial agent in solution, 2) its concentration in body fluids or tissues, and 3) the
sensitivity of a given microorganism to known concentrations of the drug. Determination
Chapter I
30
of the concentration may be undertaken by one of two principal methods viz. dilution or
diffusion method.
2.3.4.2 Principle
Antibacterial study was carried out for the synthesized DHPMs (4a-i) by disc
diffusion method against ATCC gram positive and gram negative bacterial strains
(Cruickshank et al., 1975).
2.3.4.3 Materials requirement
• The gram positive organism used for this study was Staphylococcus aureus
(ATCC 12600) and the gram negative organism was Escherichia coli (ATCC
11775) (The strains were received from Department of Veterinary Microbiology,
Madras Veterinary College, Chennai-600 007)
• The medium Tryptose Soy Agar (TSA) powder (HiMedia, Mumbai) was used at 4
g /100 ml to prepare solid agar plates and was used for both Gram positive and
Gram negative bacteria.
2.3.4.4 Method
1. The sterile discs (6 mm diameter, HiMedia) containing compounds were prepared
by dissolving the compounds in DMSO at 10 mg/ml concentration. From this
stock 100 µg concentration per disc of each compound were prepared and kept
ready.
2. Overnight cultures of each organism in Tryptose Soy broth was prepared to use in
this study.
Chapter I
31
3. The cultures were spread on the solid agar plates with a sterile cotton swab and
allowed to dry. Then the compound loaded discs were placed with equal distance
on the organism inoculated plates.
4. The plates were incubated at 37ºC for 24 h and the readings were taken by
measuring the diameter of the discs with zone of inhibition in the plates.
5. Student’s‘t’ test was used for statistical analysis. P values < 0.001 and P<0.01 were
considered to be statistically significant.
Chapter I
32
2.4 SPECTRAL DATA
2.4.1 Ethyl 4-(3-hydroxyphenyl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one-5-
carboxylate (4a)
Yield: 84%. Mp: 164–166◦C. IR (KBr): 3518, 3351, 3243, 1722, 1639 cm-1. 1H
NMR δ: 1.07 (t, J = 6.9 Hz, 3H), 2.27 (s, 3H), 3.95 (q,J = 6.9 Hz, 2H), 5.03 (s, 1H,
CH(4)), 6.58 (d, J = 8.6 Hz,1H), 6.63 (d, J = 7.45 Hz, 2H), 7.04 (t, J = 8 Hz, 1H), 7.66
(s,1H, NH(3)), 9.13 (s, 1H, NH(1)), 9.34 (s, 1H, Ar-OH).13C NMR δ: 14.6, 18.3, 54.3,
59.7, 99.9, 113.6, 114.7,117.4, 129.8, 146.8, 148.6, 152.8, 157.9, 165.9 MS (EI, m/
z): 276 (M+); Anal. Calcd for C14H16N2O4: C, 60.86; H, 5.84; N, 10.14. Found: C, 60.81;
H, 5.78; N, 10.06.
2.4.2 Ethyl 4-(isopropyl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one-5-carboxylate
(4b)
Yield: 88%. Mp: 192–193 ◦C. IR(KBr): 3234, 3102, 1692, 1645 cm-1. 1H NMR δ: 0.74
(d,J = 6.8 Hz, 3H), 0.82 (d, J = 6.9 Hz, 3H), 1.19 (t,J = 7.1 Hz, 3H), 1.68 (m, 1H), 2.18 (s,
3H), 3.96 (t,J = 3.6 Hz, 1H), 4.04 (m, 2H), 7.26 (s, 1H), 8.86 (s, 1H).13C NMR δ: 14.2,
16.0, 17.7, 18.5, 34.6, 55.5, 58.1, 98.2,148.4, 153.2, 165.8. MS (EI, m/z): 226 (M+); Anal.
Calcd for C11H18N2 O3: C, 58.39; H,8.02; N, 12.38. Found: C, 58.40; H, 8.00; N, 12.45.
2.4.3 Phenyl 4-(3-hydroxyphenyl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one-5-
carboxylate (4c)
Yield: 85%. Mp: 170–172 ◦C. IR (KBr): 3365, 2937, 2369, 1716, 1573,
1508 cm-1. 1H NMR δ: 2.28 (s, 3H), 5.22 (s, 1H), 6.79 (s, 1H), 6.85 (d, J = 8.6 Hz,1H),
6.88 (d, J = 7.2 Hz, 1H), 6.96 (t, J = 7.6 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 7.1 (d,
Chapter I
33
J = 6.8 Hz, 1H), 7.56 (s, 1H, NH(3)), 9.05 (s, 1H, NH(1)), 9.25 (s, 1H). 13C NMR δ: 14.5,
52.6, 113.5, 113.9, 116.5,119.7, 121.8, 125.2, 127.6, 130.2, 134.1, 148.0,
153.5, 153.6, 158.2, 170.5.MS (EI, m/z): 324 (M+); Anal. Calcd for C18H16N2O4: C,
66.66; H,4.97; N, 8.64. Found: C, 66.82; H, 4.85; N, 8.56.
2.4.4 Ethyl 4-(4-hydroxyphenyl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one-5-
carboxylate (4d)
Yield: 88%. Mp: 228–229 ◦C. IR (KBr): 3410, 3256, 2990, 1719, 1689 cm-1. 1H NMR:
δ:1.12 (t, 3H, J = 7.5 Hz), 2.22 (s, 3H), 4.03 (q, 2H, J = 7.5 Hz), 5.12 (s, 1H), 6.70 (d, 2H,
J = 9 Hz), 7.10 (d, 2H, J = 9.0 Hz), 7.5 (s, 1H, NH(3)), 9.01 (s, 1H, NH(1)), 9.20 (s, 1H,
Ar-OH). 13C NMR δ: 15.04, 17.76, 53.54, 58.76, 99.73, 114.58, 127.19, 136.54, 147.25,
152.30, 156.19 and 165.24. MS (EI, m/z): 276 (M+). Anal. Calcd for
C14H16N2O4: C, 60.86; H, 5.84; N, 10.14. Found: C, 60.11; H, 5.80; N, 10.42.
2.4.5 Ethyl 4-(4-methoxyphenyl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one-5-
carboxylate (4e)
Yield: 84%. Mp: 199– 200 ◦C. IR (KBr): 3303, 3050, 1710, 1647 cm-1. 1H NMR δ: 1.08
(t, 3H, J = 7.5 Hz), 2.24 (s, 3H), 3.71 (s, 3H, Ar-OMe), 3.97 (q, 2H, J = 7.5 Hz), 5.23 (d,
1H, J = 2.7 Hz), 6.88 (d, 2H, J = 8.58 Hz, Ar-H), 7.13 (d, 2H,J = 8.58 Hz, Ar-H), 7.67 (s,
1H, NH(3)), 9.15 (s, 1H, NH(1)). 13C NMR δ: 14.03, 18.32, 53.88, 55.61, 59.73, 100.13,
114.26, 127.96, 137.60, 148.58, 152.73, 159.00, 165.94. MS (EI, m/z): 290 (M+). Anal.
Calcd for C15H18N2O4: C, 62.08; H, 6.25; N, 9.65. Found: C, 61.93; H, 6.19; N, 13.87.
Chapter I
34
2.4.6 Ethyl 4-(4-(1,1’-biphenyl))-6-methyl-3,4-dihydropyrimidin- 2(1H)-one-5-
carboxylate (4f)
Yield: 70%. Mp: 212–214 ◦C. IR (KBr): 3224, 3105, 1692, 1639 cm-1. 1H NMR δ: 1.04
(t, J = 7.45 Hz, 3H), 2.21 (s, 3H), 3.82 (q, J = 7.45 Hz, 2H), 5.43 (d, 1H, J = 3.5 Hz), 7.26
(m, 3H), 7.38 (t, J = 7.45 Hz, 2H), 7.54 (t, J = 8.1 Hz, 4H), 7.71 (s, 1H, NH(3)), 9.12 (s,
1H, NH(1)). 13C NMR: δ 14.53, 18.27, 54.11, 60.05, 99.94, 127.09, 127.29, 127.41,
128.01,129.52, 139.83, 140.19, 144.23, 148.88, 152.88, 166.06. MS
(EI, m/z): 336 (M+). Anal. Calcd for C20H20N2O3: C, 71.41; H, 5.99; N, 8.33. Found: C,
71.33; H, 5.91; N, 8.37.
2.4.7 Ethyl 4-(2-nitrophenyl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one-5-
carboxylate (4g)
Yield: 90%. Mp: 213– 215 ◦C. IR (KBr): 3112, 1693, 1645, 1524, 1340 cm-1. 1H NMR
δ: 0.93 (t, 3H, J = 7.6 Hz), 2.29 (s, 3H), 3.83 (q, 2H, J = 7.6 Hz), 5.83 (s, 1H), 7.48–7.50
(m, 2H), 7.66 (s, 1H, NH (3)), 7.70 (d, 2H, J = 9 Hz), 9.36 (s, 1H, NH (1)). 13C
NMR δ: 13.58, 17.49, 49.20, 58.97, 97.92, 123.67, 128.45, 128.87, 133.85, 139.13,
146.62, 149.42, 150.60, 165.32. MS (EI, m/z): 305 (M+). Anal. Calcd for C14H15N3O5: C,
55.08; H, 4.95; N, 13.76. Found: C, 55.10; H, 4.91; N, 13.87.
2.4.8 Methyl 4-(2-chlorophenyl)-6-methyl-3,4-dihydropyrimidin-2(1H)-one-5-
carboxylate (4h)
Yield: 89%. Mp: 224–226 ◦C; IR: 3310, 3250, 1701, 1645 cm-1. 1H NMR (DMSO-d6) δ:
2.29 (s, 3H), 3.68 (s, 3H), 5.31 (s, 1H), 7.30–7.52 (m, 4H), 7.53 (s, 1H, NH(3)), 9.31 (s,
1H, NH(1)). 13C NMR δ: 17.85, 54.41, 59.23, 101.83, 126.13, 127.23, 127.51, 128.22,
Chapter I
35
140.11, 144.54, 150.21, 152.13 and 165.58. MS (EI, m/z): 280 (M+) 282 (M+2) Anal.
Calcd for C13H13ClN2O3 C, 55.62; H, 4.67; N, 9.98. Found: C, 55.43; H 4.72; N, 9.97.
2.4.9 Ethyl 4-(2-chlorophenyl)-6-methyl-3,4-dihydropyrimidin-2(1(H)-one-5-
carboxylate (4i)
Yield: 91%. Mp: 213–215 ◦C. IR (KBr): 3363, 3225, 3100, 1690, 1650 cm-1. 1H NMR δ:
1.03 (t, 3H, J = 7.5 Hz), 2.45 (s, 3H), 3.98 (q, 2H, J = 7.5 Hz), 5.64 (s, 1H, NH(1)), 5.88
(d, 1H, J = 2.4 Hz),7.20–7.24 (m, 3H), 7.36–7.39 (m, 1H), 7.87 (s, 1H, NH(1)).
13C NMR δ: 15.21, 17.88, 53.17, 58.03, 103.13, 127.23, 128.14, 129.32, 136.54, 140.11,
147.21, 152.37, 156.39 and 165.93. MS (EI, m/z): 294 (M+) 296 (M+2) Anal. Calcd for
C14H15ClN2O3: C, 57.05; H, 5.13; N, 9.50. Found: C, 57.02; H, 5.10; N, 9.52 .
Chapter I
36
2.5 RESULTS AND DISCUSSION
DHPMs scaffolds are exhibiting wide range of biological and medicinal activities.
DHPMs have emerged as second generation (Rovnyak et al., 1995) substitute for the
well-established calcium channel antagonists, Hantzsch dihydropyridines (DHPs). As
early as 1930s, its simple derivative, 4-chlorophenyl-2-thio-DHPM was patented as an
agent for the protection of wool against moths (Ertan et al., 1944). Later, the interest has
focused on the antiviral activity of Biginelli compounds, eventually leading to the
excellent antiviral agent, nitractin (Hurst and Ann, 1962). The very same compound also
exhibits modest antibacterial activity.
2.5.1 Synthesis of 4-(substituted)-3,4-dihydropyrimidinones
The synthetic pathway employed in the preparation of 4-substituted-3,4-
dihydropyrimidinones is outlined in Scheme 1. DHPMs were prepared readily by heating
1,3-dicarbonyl compounds 1, urea 3, and aromatic aldehydes 2 in acetic acid under
microwave irradiation conditions. The formation of the products was monitored through
TLC and irradiation was continued for appropriate time until completion of the reaction.
The structures of the compounds 4a–i were confirmed by spectral (Spectrum 1.1 and
1.2), elemental analyses, and comparison with the available literature data (Ma et al.,
2000; Shanmugam and Perumal, 2003; Shanmugam et al., 2003).
Chapter I
37
R''
OCO2R NH2 NH2
O
N
NH
CH3
OH
R''
RO
O R'
MWI, 7 minR'CHOAcetic acid
+ +
4a) R = Et, R' = 3-HOC6H4, R'' = CH34b) R = Et, R' = isopropyl, R'' = CH34c) R = Ph, R' = 3-HOC6H4, R'' = CH34d) R = Et, R' = 4-HOC6H4, R'' = CH34e) R = Et, R' = 4-MeOC6H4, R'' = CH3
4f) R = Et, R' = biphenyl, R'' = CH34g) R = Et, R' = 2- O2NC6H4, R'' =CH34h) R = Me, R' = 2-ClC6H4, R'' = CH34i) R = Et, R' = 2-ClC6H4, R'' = CH3
1 2 3 4 (a-i)
Scheme 1
The formation of 4 was confirmed by 1H NMR spectra (Spectrum 1.1), which
displays the CH (4) peaks at δ 5.0-6.0. IR spectra confirm the presence of the carbonyl
peaks of amide and ester at 1650 and 1690 cm-1 respectively and intense NH absorptions
at 3400-3200 cm-1. Mass spectra show the base peak corresponding to (M+-Ar) at 183
(for ethyl acetoacetate series), and elemental analysis additionally corroborated the
results. Tabulation of results are depicted below (Table 1.1).
Chapter I
38
Table1.1 Synthesis of 3,4-Dihydropyrimidin-2(1H)-ones
Compound No.
R R’ R” Yield (%)
4a Ethyl 3-hydroxy phenyl
Methyl 84
4b Ethyl Isopropyl Methyl 88
4c Phenyl 3-hydroxy phenyl
Methyl 85
4d Ethyl 4-hydroxy phenyl
Methyl 88
4e Ethyl 4-methoxy phenyl
Methyl 84
4f Ethyl Biphenyl Methyl 70
4g Ethyl 2-nitro phenyl Methyl 90
4h Methyl 2-chloro phenyl Methyl 89
4i Ethyl 2-chloro phenyl Methyl 91
2.5.2 Cardiovascular activity Cardiovascular diseases refer to the class of diseases that involve the heart and /
or blood vessels. Over the last several decades, clinical research on cardiovascular
disease has advanced and improved tremendously. This has resulted in prevention as well
as treatment of America’s number one killer of men and women of all races. The
American Heart Association estimates that more than 50 % of deaths are related to some
form of cardiovascular diseases. (Ahluwalia and Madhu Chopra, 2008) The
cardiovascular effects of DHPMs were evaluated on an isolated perfused frog heart at
various dose levels and compared with the activity of digoxin under identical
experimental conditions and the recordings were noted. To elucidate the mechanism of
action, the interaction of selected compounds with β-blocker and calcium channel blocker
was also investigated (Figs.1.6–1.14) and the results are presented in the Table 1.2-1.4.
Chapter I
39
In the present investigation compounds 4a–d showed dose-dependent increase in
force of contraction (positive inotropic action), decrease in rate of contraction (negative
chronotropic action), and an increase in cardiac output. Among these compounds 4d was
more potent than digoxin. This may be contributed by the more potent calcium channel
agonistic S enantiomer (Kappe, 2000b) in the racemic mixture of R and S. Change of
‘ethyl’ (compound 4a) by ‘phenyl’ (compound 4c) substitution in the ester portion of the
DHPM does not have marked effect on cardiovascular activity. Compound 4e showed
negative chronotropic action, increase in cardiac output, positive inotropic action, and
was not dose-dependent. Compound 4f at lower dose levels (at 5 and 50 μg/ml) did not
show enhanced activity but at higher dose levels (at 500 μg/ml and 1 mg/ml) showed
comparable positive inotropic action and an increase in cardiac output. Compound 4g
showed no change in rate, force of contraction of the heart and cardiac output as expected
(Kappe, 2000b) .Compounds 4h and 4i were found to exhibit negative inotropic,
chronotropic action, and decrease in cardiac output (Kappe, 2000b). Further 4h and 4i
blocked the effect of adrenalin (5 μg/ml), thereby showing β-adrenergic blocking activity.
The positive inotropic action of the compounds (4a–f) was not blocked by
metoprolol (a cardioselective β-adrenergic blocker) but significantly blocked by the
calcium channel blocker—verapamil. Since verapamil blocks the cardiotonic action of
the compounds (4a–f), these compounds might have produced their action by opening the
voltage sensitive slow Ca2+ channel.
Chapter I
40
2.5.3 Antibacterial activity Compounds 4a-i was tested against Escherichia coli (ATCC 11775) and
Staphylococcus aureus (ATCC 12600) at 100 µg concentration. Compounds 4e, 4h and
4i showed significant antibacterial activity. Compounds 4a, 4b, 4c, 4d, 4f and 4g did not
show any activity (Table 1.5 and Fig. 1.15).
2.6 SUMMARY
Chapter I
41
In summary, description of the present work on the synthesis, antibacterial and
pharmacological effect of DHPMs on frog heart. The study deals with the synthesis of
DHPMs by Biginelli reaction using aldehyde, β-keto ester and urea in the presence of
acetic acid as catalyst. The results obtained clearly indicate that the compounds 4a–f
discussed here showed good cardiotonic activity. Compounds 4h and 4i evinced β-
adrenergic receptor antagonistic activity. Compound 4d appears to be the most interesting
derivative in our series and more potent than the digoxin. It can be a better choice for the
existing cardiotonic drugs in the treatment of congestive heart failure and to confirm this,
further studies are to be carried out in other laboratory animals. Compounds 4e, 4h and 4i
showed significant antibacterial activity. This observation may promote the synthesis of
more active DHPMS in future. The development of methods for the enantioselective
synthesis of chiral DHPMs using chiral catalysts and enzymes is underway since
individual enantiomers of chiral DHPMs have opposing pharmacological effects and the
use of enantiomerically pure compounds are a requirement for improving the efficacy of
drugs of this type.
Chapter I
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Chapter I
Spectrum 1.1 1H NMR of DHPM (4e)
Chapter I
Spectrum 1.2 13C NMR of DHPM (4e)
Chapter I
Table 1.2 The effects of dihydropyrimidinone derivatives (Force of Contraction) on
frog heart Entry
Controla 5 μg 50 μg 100 μg 500 μg 1 mg Metoprolol Verapamil
FC (mm) FC (mm) FC (mm) FC (mm) FC (mm) FC (mm) FC (mm) FC (mm)
4a 13 ± 0.58 20 ± 0.58 25 ± 0.58 28 ± 0.58 30 ± 0.82 31 ± 0.58 31 ± 0.58 03 ± 0.58
4b 12 ± 0.57 24 ± 0.82 27 ± 0.58 29 ± 0.82 31 ± 0.82 33 ± 0.82 24 ± 0.82 03 ± 0.37
4c 11 ± 0.77 21 ± 0.82 24 ± 1.15 26 ± 1.63 33 ± 1.29 35 ± 1.39 33 ± 0.52 03 ± 0.26
4d 15 ± 1.31 51 ± 1.63 59 ± 1.96 63±1.39 67±1.03 - 64 ± 1.63 02 ± 0.37
4e 15 ± 0.82 24 ± 0.57 24 ± 1.15 24 ± 1.15 24 ± 0.58 24 ± 1.50 24 ± 0.77 05±0.93
4f 15 ± 0.93 27 ± 0.58 27 ± 1.82 27 ± 0.58 35 ± 1.39 35 ± 1.15 35 ± 0.26 04±0.37
4g 08 ± 0.26 08 ± 0.37 08 ± 0.58 08 ± 0.26 08 ± 0.82 08 ± 0.58 - -
4h 11 ± 0.58 04 ± 0.26 03 ± 0.37 03 ± 0.58 02 ± 0.26 - - -
4i 12 ± 0.58 05 ± 0.36 01 ± 0.26 01 ± 0.37 - - - -
Digoxin 11 ± 0.26 40±1.39 47 ± 0.93 - 51 ± 1.03 - - -
a 1% Sodium carboxy methyl cellulose as control. n=6, values are mean± SEM. All compounds showed p∠ 0.01 when compared with control (one –way ANOVA) except entry 4g.
Chapter I
Fig. 1.6 The effects of dihydropyrimidinone derivatives (force of contraction) on frog heart
Chapter I
Table 1.3 The effect of dihydropyrimidinone derivatives (Heart Rate) on frog heart
Entry
Controla 5 μg 50 μg 100 μg 500 μg 1 mg Metoprolol Verapamil HR (bpm)
HR (bpm)
HR (bpm)
HR (bpm)
HR (bpm)
HR (bpm)
HR (bpm)
HR (bpm)
4a 80 ± 0.58 70 ± 0.58 68 ± 0.42 68 ± 1.16 68 ± 1.12 68 ± 1.11 68 ± 0.37 17 ± 0.37
4b 78 ± 0.58 58 ± 0.57 56 ± 0.58 57 ± 0.58 56 ± 0.57 56 ± 0.58 58 ± 0.26 21 ± 0.26
4c 78 ± 1.39 65 ± 1.65 650 ± 2.69 650 ± 0.29 65 ± 1.15 65 ± 1.65 64 ± 0.82 17 ± 0.58 4d 97 ± 1.39 87 ± 1.39 77 ± 1.51 77 ± 1.65 64 ± 0.82 - 64 ± 1.63 27 ± 1.15
4e 105 ± 0.75 94 ± 0.75 94 ± 1.12 94 ± 0.76 94 ± 1.11 94 ± 0.75 94 ± 1.16 20 ± 0.93
4f 100 ± 0.93 93 ± 1.32 94 ± 0.52 95 ± 0.73 95 ± 1.80 95 ± 1.31 95 ± 0.37 18 ± 0.82
4g 100 ± 0.37 100 ± 0.58 100 ± 0.82 100 ± 0.37 100 ± 0.26 100 ± 0.82 - -
4h 97 ± 2.88 88 ± 1.97 84 ± 0.91 78 ± 1.39 78 ± 1.63 - - -
4i 96 ± 2.06 92 ± 1.15 84 ± 1.39 78 ± 0.93 - - - -
Digoxin 97 ± 0.82 70 ± 0.76 68 ± 1.06 - 68 ± 0.93 - - -
a 1% Sodium carboxy methyl cellulose as control. n=6, values are mean± SEM. All compounds showed p∠ 0.01 when compared with control (one –way ANOVA) except entry 4g.
Chapter I
Fig. 1.7 The effects of dihydropyrimidinone derivatives (heart rate) on frog heart
Chapter I
Table 1.4 The effects of Dihydropyrimidinone derivatives (cardiac output-CO) on frog heart
Entry
Controla 5 μg 50 μg 100 μg 500 μg 1 mg Metoprolol Verapamil
COml/m
COml/m
COml/m
COml/m
COml/m
COml/m
COml/m
COml/m
4a 9 ± 0.058 10 ± .082 11 ± 0.58 11.5 ± 0.037 12 ±0.082 12 ± 0.032 11 ± 0.15 03 ± 0.037
4b 15 ± 0.82 17 ± 0.021 17 ± 1.16 18 ± 0.82 18.4 ±1.39 18.5 ± 0.37 17 ± 0.021 06 ± 0.58
4c 11 ± 0.58 12.5 ± 0.76 12 ± 0.93 12.4 ± 0.26 13.1 ±1.16 13.3 ± 0.58 12.5 ± 0.26 04 ± 0.021
4d 16 ± 0.52 20 ± 0.29 22.3 ± 0.82 23.1 ± 0.021 24.2 ±0.75 - 24± 0.82 07 ± 1.15
4e 15 ± 0.021 16 ± 1.16 16.1±0.037 16.8 ± 0.29 17 ± 1.12 17.1 ± 0.52 17 ± 0.58 07 ± 0.93
4f 19 ± 1.63 20 ± 1.11 22 ± 0.058 22 ± 0.021 23 ± 1.16 23 ± 0.58 23 ± 0.082 06 ± 0.037
4g 20 ± 0.058 20 ± 0.021 20 ± 0.58 20 ± 1.39 20 ± 0.91 20 ± 1.65 - -
4h 20 ± 0.58 11 ± 1.39 9.1 ± 0.93 8.8 ± 1.97 8.4 ±0.021 - - -
4i 18 ± 0.42 06 ± 0.76 4.5 ± 1.11 4.3 ± 0.021 - - - -
Digoxin 14 ± 1.15 18 ± 1.16 18.4 ±0.58 - 19.1 ±0.82 - - -
a 1% Sodium carboxy methyl cellulose as control. n=6, values are mean± SEM. All compounds showed p∠ 0.01 when compared with control (one –way ANOVA) except entry 4g.
Chapter I
Fig. 1.8 The effects of dihydropyrimidinone derivatives (cardiac output-CO) on frog heart
Fig. 1.9 Cardiogram of Frog for Digoxin and compound 4a
Car
diac
out
put m
l/m
Chapter I
Fig. 1.10 Cardiogram of Frog for compound 4b
Fig. 1.11 Cardiogram of Frog for compounds 4c and 4d
Chapter I
Fig. 1.12 Cardiogram of Frog for compounds 4d and 4e
Fig. 1.13 Cardiogram of Frog for compounds 4f and 4g
Chapter I
Fig. 1.14 Cardiogram of Frog for compounds 4h and 4i
Chapter I
Chapter I
Table 1.5 Antibacterial activity of DHPMs
Compound name
Zone of inhibition (mm)
Staphylococcus aureus Escherichia coli
4a - -
4b - -
4c - -
4d - -
4e 10.0** 10.5**
4f - -
4g - -
4h 13.0** 9.0*
4i 9.5** 9.0*
Control 8.0 8.0
Standard Ciprofloxacin-35** (5 μg) Gentamicin-30** (10 μg)
* P<0.01, ** P<0.001 when compared with control (student’s‘t’ – test)
Chapter I
Fig. 1.15 Antibacterial activity of DHPMs
Compounds and DMSO control tested against Staphylococcus aureus
